Molecular characterization of the RNA-protein complex directing −2/−1 programmed ribosomal frameshifting during arterivirus replicase expression
2020; Elsevier BV; Volume: 295; Issue: 52 Linguagem: Inglês
10.1074/jbc.ra120.016105
ISSN1083-351X
AutoresAnkoor Patel, Emmely E. Treffers, Markus Meier, Trushar R. Patel, Jörg Stetefeld, Eric J. Snijder, Brian L. Mark,
Tópico(s)Viral gastroenteritis research and epidemiology
ResumoProgrammed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs −1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical −1 and −2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1β (nsp1β) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1β and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate −1 and −2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1β and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1β:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1β within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1β and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1β and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism. Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs −1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical −1 and −2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1β (nsp1β) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1β and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate −1 and −2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1β and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1β:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1β within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1β and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1β and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism. RNA viruses have evolved remarkable noncanonical translational mechanisms to maximize the coding capacity of their genomes (1Firth A.E. Brierley I. Non-canonical translation in RNA viruses.J. Gen. Virol. 2012; 93 (22535777): 1385-140910.1099/vir.0.042499-0Crossref PubMed Scopus (316) Google Scholar, 2Atkins J.F. Loughran G. Bhatt P.R. Firth A.E. Baranov P.V. Ribosomal frameshifting and transcriptional slippage: from genetic steganography and cryptography to adventitious use.Nucleic Acids Res. 2016; 44: 7007-707810.1093/nar/gkw530PubMed Google Scholar), including the use of programmed ribosomal frameshifting (PRF). PRF enables the ribosome to access multiple overlapping ORFs within the viral genome (1Firth A.E. Brierley I. Non-canonical translation in RNA viruses.J. Gen. Virol. 2012; 93 (22535777): 1385-140910.1099/vir.0.042499-0Crossref PubMed Scopus (316) Google Scholar, 3Somogyi P. Jenner A.J. Brierley I. Inglis S.C. Ribosomal pausing during translation of an RNA pseudoknot.Mol. Cell Biol. 1993; 13 (8413285): 6931-694010.1128/mcb.13.11.6931Crossref PubMed Scopus (192) Google Scholar, 4Kontos H. Napthine S. Brierley I. Ribosomal pausing at a frameshifter RNA pseudoknot is sensitive to reading phase but shows little correlation with frameshift efficiency.Mol. Cell Biol. 2001; 21 (11713298): 8657-867010.1128/MCB.21.24.8657-8670.2001Crossref PubMed Scopus (115) Google Scholar), thus yielding alternative viral protein variants from what—upon cursory inspection—appears to be a single gene, allowing for the expression of partially colinear proteins with alternate C-terminal extensions and domains (1Firth A.E. Brierley I. Non-canonical translation in RNA viruses.J. Gen. Virol. 2012; 93 (22535777): 1385-140910.1099/vir.0.042499-0Crossref PubMed Scopus (316) Google Scholar, 5Brierley I. Digard P. Inglis S. Characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an RNA pseudoknot.Cell. 1989; 57 (2720781): 537-54710.1016/0092-8674(89)90124-4Abstract Full Text PDF PubMed Scopus (486) Google Scholar, 6Giedroc D. Cornish P. Frameshifting RNA pseudoknots: structure and mechanism.Virus Res. 2009; 139 (18621088): 193-20810.1016/j.virusres.2008.06.008Crossref PubMed Scopus (213) Google Scholar). The first evidence for the occurrence of PRF was discovered in Rous sarcoma virus, which produces a gag-pol fusion protein from briefly overlapping gag and pol ORFs during infection (7Jacks T. Varmus H.E. Expression of the Rous sarcoma virus pol gene by ribosomal frameshifting.Science. 1985; 230 (2416054): 1237-124210.1126/science.2416054Crossref PubMed Scopus (285) Google Scholar, 8Jacks T. Madhani H.D. Masiarz F.R. Varmus H.E. Signals for ribosomal frameshifting in the Rous sarcoma virus gag-pol region.Cell. 1988; 55 (2846182): 447-45810.1016/0092-8674(88)90031-1Abstract Full Text PDF PubMed Scopus (433) Google Scholar, 9Jacks T. Townsley K. Varmus H.E. Majors J. Two efficient ribosomal frameshifting events are required for synthesis of mouse mammary tumor virus gag-related polyproteins.Proc. Natl. Acad. Sci. U. S. A. 1987; 84 (3035577): 4298-430210.1073/pnas.84.12.4298Crossref PubMed Scopus (133) Google Scholar). This is achieved by causing the host cell ribosome to slip back one position (−1 PRF) during translation of the viral RNA genome, which occurs at a heptameric "slippery" sequence that is located 5–10 nucleotides upstream of an RNA structural element (stem-loop or pseudoknot) (7Jacks T. Varmus H.E. Expression of the Rous sarcoma virus pol gene by ribosomal frameshifting.Science. 1985; 230 (2416054): 1237-124210.1126/science.2416054Crossref PubMed Scopus (285) Google Scholar, 8Jacks T. Madhani H.D. Masiarz F.R. Varmus H.E. Signals for ribosomal frameshifting in the Rous sarcoma virus gag-pol region.Cell. 1988; 55 (2846182): 447-45810.1016/0092-8674(88)90031-1Abstract Full Text PDF PubMed Scopus (433) Google Scholar, 9Jacks T. Townsley K. Varmus H.E. Majors J. Two efficient ribosomal frameshifting events are required for synthesis of mouse mammary tumor virus gag-related polyproteins.Proc. Natl. Acad. Sci. U. S. A. 1987; 84 (3035577): 4298-430210.1073/pnas.84.12.4298Crossref PubMed Scopus (133) Google Scholar). Encountering this RNA structure causes the ribosome to pause and "slip" on the slippery sequence, resulting in a −1 frameshift that opens access to an alternate reading frame (10Tu C. Tzeng T.H. Bruenn J.A. Ribosomal movement impeded at a pseudoknot required for frameshifting.Proc. Natl. Acad. Sci. U. S. A. 1992; 89 (1528874): 8636-864010.1073/pnas.89.18.8636Crossref PubMed Scopus (142) Google Scholar). The frequency of frameshifting events differs per virus and presumably controls the stoichiometry of certain viral proteins (1Firth A.E. Brierley I. Non-canonical translation in RNA viruses.J. Gen. Virol. 2012; 93 (22535777): 1385-140910.1099/vir.0.042499-0Crossref PubMed Scopus (316) Google Scholar). Members of the order Nidovirales (including among others the families Arteriviridae and Coronaviridae) encode two large replicase polyproteins, pp1a and pp1ab, which are post-translationally cleaved to yield 12–16 mature nonstructural proteins (nsps) (11Snijder E.J. Kikkert M. Fang Y. Arterivirus molecular biology and pathogenesis.J. Gen. Virol. 2013; 94 (23939974): 2141-216310.1099/vir.0.056341-0Crossref PubMed Scopus (296) Google Scholar). Expression of pp1ab depends on a pseudoknot-stimulated −1 PRF event to occur in the short ORF1a/ORF1b overlap region (5Brierley I. Digard P. Inglis S. Characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an RNA pseudoknot.Cell. 1989; 57 (2720781): 537-54710.1016/0092-8674(89)90124-4Abstract Full Text PDF PubMed Scopus (486) Google Scholar, 12den Boon J.A. Snijder E.J. Chirnside E.D. de Vries A.A. Horzinek M.C. Spaan W.J. Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily.J. Virol. 1991; (1851863)10.1128/jvi.65.6.2910-2920.1991Crossref PubMed Google Scholar). Next to this well-characterized −1 PRF event, most members of the arterivirus family also employ a more unusual −2 PRF mechanism. For example, in porcine reproductive and respiratory syndrome virus (PRRSV) and simian hemorrhagic fever virus (SHFV), −1 and −2 PRF events were shown to occur at the same site in the nsp2-coding region of ORF1a, yielding two nsp2 variants. In the case of PRRSV (Fig. 1A), these products are either truncated compared with full-length nsp2 (nsp2N, resulting from −1 PRF) or contain an alternative C-terminal domain (nsp2TF, resulting form −2 PRF) and were implicated in suppressing host innate immune responses (Fig. 1A) (13Li Y. Treffers E.E. Napthine S. Tas A. Zhu L. Sun Z. Bell S. Mark B.L. Veelen P.A. van Hemert M.J. Firth A.E. Brierley I. Snijder E.J. Fang Y. Transactivation of programmed ribosomal frameshifting by a viral protein.Proc. Natl. Acad. Sci. U. S. A. 2014; 111 (24825891): E2172-E218110.1073/pnas.1321930111Crossref PubMed Scopus (103) Google Scholar, 14Fang Y. Treffers E.E. Li Y. Tas A. Sun Z. van der Meer Y. de Ru A.H. van Veelen P.A. Atkins J.F. Snijder E.J. Firth A.E. Efficient -2 frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein.Proc. Natl. Acad. Sci. U. S. A. 2012; 109 (23043113): E2920-E292810.1073/pnas.1211145109Crossref PubMed Scopus (192) Google Scholar, 15Li Y. Shang P. Shyu D. Carrillo C. Naraghi-Arani P. Jaing C.J. Renukaradhya G.J. Firth A.E. Snijder E.J. Fang Y. Nonstructural proteins nsp2TF and nsp2N of porcine reproductive and respiratory syndrome virus (PRRSV) play important roles in suppressing host innate immune responses.Virology. 2018; 517 (29325778): 164-17610.1016/j.virol.2017.12.017Crossref PubMed Scopus (23) Google Scholar, 16Li Y. Firth A.E. Brierley I. Cai Y. Napthine S. Wang T. Yan X. Kuhn J.H. Fang Y. Programmed −2/−1 ribosomal frameshifting in Simarteriviruses: an evolutionarily conserved mechanism.J. Virol. 2019; 93 (31167906): 1-2110.1128/jvi.00370-19Crossref Scopus (12) Google Scholar). Interestingly, whereas a characteristic slippery sequence is present in the region of the PRRSV genome where these frameshifts occur, no discernible RNA secondary structural element could be predicted (14Fang Y. Treffers E.E. Li Y. Tas A. Sun Z. van der Meer Y. de Ru A.H. van Veelen P.A. Atkins J.F. Snijder E.J. Firth A.E. Efficient -2 frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein.Proc. Natl. Acad. Sci. U. S. A. 2012; 109 (23043113): E2920-E292810.1073/pnas.1211145109Crossref PubMed Scopus (192) Google Scholar). However, a highly conserved C-rich motif (CCCANCUCC, or similar) is found 11 nt downstream of the slippery sequence shift site in studied PRRSV isolates (Fig. 1B), which suggested that a novel transactivating mechanism facilitates PRF at this position as opposed to a ribosomal pausing mechanism that is usually induced by an RNA tertiary structural element (13Li Y. Treffers E.E. Napthine S. Tas A. Zhu L. Sun Z. Bell S. Mark B.L. Veelen P.A. van Hemert M.J. Firth A.E. Brierley I. Snijder E.J. Fang Y. Transactivation of programmed ribosomal frameshifting by a viral protein.Proc. Natl. Acad. Sci. U. S. A. 2014; 111 (24825891): E2172-E218110.1073/pnas.1321930111Crossref PubMed Scopus (103) Google Scholar, 17Napthine S. Treffers E.E. Bell S. Goodfellow I. Fang Y. Firth A.E. Snijder E.J. Brierley I. A novel role for poly(C) binding proteins in programmed ribosomal frameshifting.Nucleic Acids Res. 2016; 44 (27257056): 5491-550310.1093/nar/gkw480Crossref PubMed Scopus (29) Google Scholar). Indeed, two trans-acting elements subsequently were shown to control −1 and −2 PRF in PRRSV: the PRRSV protein nsp1β and the host cell protein poly(C)-binding protein 1 or 2 (PCBP1 or -2) (13Li Y. Treffers E.E. Napthine S. Tas A. Zhu L. Sun Z. Bell S. Mark B.L. Veelen P.A. van Hemert M.J. Firth A.E. Brierley I. Snijder E.J. Fang Y. Transactivation of programmed ribosomal frameshifting by a viral protein.Proc. Natl. Acad. Sci. U. S. A. 2014; 111 (24825891): E2172-E218110.1073/pnas.1321930111Crossref PubMed Scopus (103) Google Scholar, 14Fang Y. Treffers E.E. Li Y. Tas A. Sun Z. van der Meer Y. de Ru A.H. van Veelen P.A. Atkins J.F. Snijder E.J. Firth A.E. Efficient -2 frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein.Proc. Natl. Acad. Sci. U. S. A. 2012; 109 (23043113): E2920-E292810.1073/pnas.1211145109Crossref PubMed Scopus (192) Google Scholar, 17Napthine S. Treffers E.E. Bell S. Goodfellow I. Fang Y. Firth A.E. Snijder E.J. Brierley I. A novel role for poly(C) binding proteins in programmed ribosomal frameshifting.Nucleic Acids Res. 2016; 44 (27257056): 5491-550310.1093/nar/gkw480Crossref PubMed Scopus (29) Google Scholar). The two proteins interact with each other and with the viral RNA genome to induce −1/−2 PRF in the nsp2-coding region of ORF1a. Although PCBP2 and nsp1β had previously been shown to interact with each other (18Beura L.K. Dinh P.X. Osorio F.A. Pattnaik A.K. Cellular poly(C) binding proteins 1 and 2 interact with porcine reproductive and respiratory syndrome virus nonstructural protein 1β and support viral replication.J. Virol. 2011; 85 (21976648): 12939-1294910.1128/JVI.05177-11Crossref PubMed Scopus (46) Google Scholar), the significance of this interaction for efficient PRF has only recently been discovered (13Li Y. Treffers E.E. Napthine S. Tas A. Zhu L. Sun Z. Bell S. Mark B.L. Veelen P.A. van Hemert M.J. Firth A.E. Brierley I. Snijder E.J. Fang Y. Transactivation of programmed ribosomal frameshifting by a viral protein.Proc. Natl. Acad. Sci. U. S. A. 2014; 111 (24825891): E2172-E218110.1073/pnas.1321930111Crossref PubMed Scopus (103) Google Scholar, 17Napthine S. Treffers E.E. Bell S. Goodfellow I. Fang Y. Firth A.E. Snijder E.J. Brierley I. A novel role for poly(C) binding proteins in programmed ribosomal frameshifting.Nucleic Acids Res. 2016; 44 (27257056): 5491-550310.1093/nar/gkw480Crossref PubMed Scopus (29) Google Scholar). PRRSV remains the most economically important viral disease in the swine industry (19Lunney J.K. Fang Y. Ladinig A. Chen N. Li Y. Rowland B. Renukaradhya G.J. Porcine reproductive and respiratory syndrome Virus (PRRSV): pathogenesis and interaction with the immune system.Annu. Rev. Anim. Biosci. 2016; 4 (26646630): 129-15410.1146/annurev-animal-022114-111025Crossref PubMed Scopus (345) Google Scholar), and its high pathogenicity may be due, in part, to the immune evasion mechanisms it employs during infection (11Snijder E.J. Kikkert M. Fang Y. Arterivirus molecular biology and pathogenesis.J. Gen. Virol. 2013; 94 (23939974): 2141-216310.1099/vir.0.056341-0Crossref PubMed Scopus (296) Google Scholar). Consequently, the further dissection of its molecular biology and gene expression mechanisms is highly relevant for efforts to improve PRRSV vaccines, including those based on attenuation by targeted engineering of the viral genome (20Hu J. Zhang C. Porcine reproductive and respiratory syndrome virus vaccines: current status and strategies to a universal vaccine.Transbound. Emerg. Dis. 2014; 61 (23343057): 109-12010.1111/tbed.12016Crossref PubMed Scopus (50) Google Scholar). Despite its importance to PRRSV replication, the biochemistry and structural biology of the interactions between nsp1β, PCBP1 or -2, and the PRRSV RNA genome have not been explored. Here we provide structural and functional insights into the quaternary complex between nsp1β:PCBP2 and viral RNA that controls PRF. Site-directed mutations in both nsp1β and PCBP2 pinpointed key residues needed for complex formation with the RNA genome. Nsp1β mutagenesis was also used to identify residues essential to stimulate PRF as well as residues involved in the evasion of innate immune responses. Whereas we found nsp1β and PCBP2 to be unstable on their own, combining the proteins with viral RNA containing the putative slippery sequence and C-rich motif resulted in a highly stable complex that we could study by analytical ultracentrifugation and small-angle X-ray scattering. Our study provides detailed molecular insights into a novel PRF-directing mechanism employing two protein transactivators interacting with the PRRSV genome to expand its coding capacity. To gain insights into how nsp1β and PCBP2 interact with specific sequences in the PRRSV RNA genome to induce frameshifting, we first developed a robust expression and purification scheme for the proteins. Of the PRRSV isolates we tested, nsp1β from isolate SD01-08, a low-virulence European isolate from species Betaarterivirus suid 1 (formerly type I PRRSV), was found to be the most amenable to overexpression and purification using Escherichia coli BL21(DE3) as an expression host (Fig. 2A). Whereas we tried to also express and purify the structurally characterized nsp1β from the highly pathogenic North American PRRSV isolate XH-GD (PDB code 3MTV) (21Sattler T. Pikalo J. Wodak E. Revilla-Fernández S. Steinrigl A. Bagó Z. Entenfellner F. Claude J.B. Pez F. Francillette M. Schmoll F. Efficacy of live attenuated porcine reproductive and respiratory syndrome virus 2 strains to protect pigs from challenge with a heterologous Vietnamese PRRSV 2 field strain.BMC Vet. Res. 2018; 14 (29673363): 1-1010.1186/s12917-018-1451-yCrossref PubMed Scopus (2) Google Scholar, 22Xue F. Sun Y. Yan L. Zhao C. Chen J. Bartlam M. Li X. Lou Z. Rao Z. The crystal structure of porcine reproductive and respiratory syndrome virus nonstructural protein Nsp1β reveals a novel metal-dependent nuclease.J. Virol. 2010; 84 (20410261): 6461-647110.1128/jvi.00301-10Crossref PubMed Scopus (41) Google Scholar) from species Betaarterivirus suid 2 (formerly type 2 PRRSV), the attempt failed due to the protein's instability in solution, even at low concentrations. Full-length human PCBP2 was also recombinantly expressed in E. coli BL21(DE3) (Fig. 2A), and whereas a 3D structure of the complete protein has not been determined, structures of its three nucleic acid–binding domains are available (K homology domains; KH1 (23Du Z. Lee J.K. Fenn S. Tjhen R. Stroud R.M. James T.L. X-ray crystallographic and NMR studies of protein-protein and protein-nucleic acid interactions involving the KH domains from human poly(C)-binding protein-2.RNA. 2007; 13 (17526645): 1043-105110.1261/rna.410107Crossref PubMed Scopus (39) Google Scholar), KH2 (23Du Z. Lee J.K. Fenn S. Tjhen R. Stroud R.M. James T.L. X-ray crystallographic and NMR studies of protein-protein and protein-nucleic acid interactions involving the KH domains from human poly(C)-binding protein-2.RNA. 2007; 13 (17526645): 1043-105110.1261/rna.410107Crossref PubMed Scopus (39) Google Scholar, 24Du Z. Fenn S. Tjhen R. James T.L. Structure of a construct of a human poly(C)-binding protein containing the first and second KH domains reveals insights into its regulatory mechanisms.J. Biol. Chem. 2008; 283 (18701464): 28757-2876610.1074/jbc.M803046200Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), and KH3 (25Fenn S. Du Z. Lee J.K. Tjhen R. Stroud R.M. James T.L. Crystal structure of the third KH domain of human poly (C) -binding protein-2 in complex with a C-rich strand of human telomeric DNA at 1. 6 Å resolution.Nucleic Acids Res. 2007; 35 (17426136): 2651-266010.1093/nar/gkm139Crossref PubMed Scopus (29) Google Scholar)) (Fig. 3A).Figure 3Structure-guided mutational analysis of PCBP2 and nsp1β binding to PRRSV RNA. A, probing nucleic acid interaction sites of PCBP2. A schematic of full-length PCBP2 showing KH1 (gray), KH2 (green), and KH3 (teal) domains with accompanying three-dimensional structures (PDB entries 2P2R (26Du Z. Lee J.K. Tjhen R. Li S. Pan H. Stroud R.M. James T.L. Crystal structure of the first KH domain of human poly(C)-binding protein-2 in complex with a C-rich strand of human telomeric DNA at 1.7 Å.J. Biol. Chem. 2005; 280 (16186123): 38823-3883010.1074/jbc.M508183200Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), 2JZX (24Du Z. Fenn S. Tjhen R. James T.L. Structure of a construct of a human poly(C)-binding protein containing the first and second KH domains reveals insights into its regulatory mechanisms.J. Biol. Chem. 2008; 283 (18701464): 28757-2876610.1074/jbc.M803046200Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), and 2PQU (25Fenn S. Du Z. Lee J.K. Tjhen R. Stroud R.M. James T.L. Crystal structure of the third KH domain of human poly (C) -binding protein-2 in complex with a C-rich strand of human telomeric DNA at 1. 6 Å resolution.Nucleic Acids Res. 2007; 35 (17426136): 2651-266010.1093/nar/gkm139Crossref PubMed Scopus (29) Google Scholar) for DNA-bound KH1, KH1-KH2 fusion, and DNA-bound KH3, respectively). The KH1 guanidino groups of Arg40 and Arg57 appear to hydrogen-bond with the keto group of a cytosine nucleobase, whereas the side chain of Asn325 in KH3 is within hydrogen-bonding distance of an adenine nucleobase. Whereas the published structure has the amino group of Asn325 interacting with adenine, it is more likely that the carboxamide is rotated 180° to allow the carbonyl group to interact with the base instead. B, probing PRRSV nsp1β interactions with nucleic acid. Shown is a schematic of nsp1β from PRRSV strain XH-GD (PDB entry 3MTV (22Xue F. Sun Y. Yan L. Zhao C. Chen J. Bartlam M. Li X. Lou Z. Rao Z. The crystal structure of porcine reproductive and respiratory syndrome virus nonstructural protein Nsp1β reveals a novel metal-dependent nuclease.J. Virol. 2010; 84 (20410261): 6461-647110.1128/jvi.00301-10Crossref PubMed Scopus (41) Google Scholar)) with the putative RNA-binding motif (14Fang Y. Treffers E.E. Li Y. Tas A. Sun Z. van der Meer Y. de Ru A.H. van Veelen P.A. Atkins J.F. Snijder E.J. Firth A.E. Efficient -2 frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein.Proc. Natl. Acad. Sci. U. S. A. 2012; 109 (23043113): E2920-E292810.1073/pnas.1211145109Crossref PubMed Scopus (192) Google Scholar) and residues Tyr131 and Arg135 shown in purple. Figures were generated using PyMOL (27DeLano W.L. The PyMOL Molecular Graphics System. Schrödinger, LLC, New York2015Google Scholar). C and D, EMSAs performed with a 20 μm concentration of the 34-nt ssRNA (Fig. 1B). In C, WT nsp1β and two mutants (Y131A and R135A) were combined with PCBP2 and the ssRNA probe. In D, WT PCBP2 and three mutants (single mutant (N325D), double mutant (N325D/R40A), and triple mutant (N325D/R40A/R57A)) were combined with nsp1β and the ssRNA probe. Molar excess of each protein is listed below each well compared with the probe.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although nsp1β and PCBP2 could be overexpressed as soluble proteins in E. coli, both were prone to aggregation and had low solubility during purification, which prompted us to identify optimal buffer systems for the proteins. This was determined empirically by screening 96 buffer conditions (Hampton Research) to find conditions that increase the thermal stability of the proteins. Increased SYPRO Orange (Sigma–Aldrich) fluorescence arising from protein unfolding was used to analyze protein denaturation curves as described (28Ericsson U.B. Hallberg B.M. DeTitta G.T. Dekker N. Nordlund P. Thermofluor-based high-throughput stability optimization of proteins for structural studies.Anal. Biochem. 2006; 357 (16962548): 289-29810.1016/j.ab.2006.07.027Crossref PubMed Scopus (667) Google Scholar). A buffer that was found to enhance the thermal stability of both proteins consisted of 1× PBS (pH 7.4), 300 mm NaCl, 100 mm KCl, and 5% glycerol. Further, because nsp1β is cysteine-rich, DTT was added to a final concentration of 2 mm to avoid cysteine oxidation. Using this optimized buffer system, we were ultimately able to isolate each protein to high purity (Fig. 2A), although concentrating either protein to above 1 mg/ml invariably led to aggregation. Nevertheless, they were stable and monodisperse at concentrations needed for nucleic acid interaction studies by electrophoretic mobility shift assays (EMSAs; Fig. 2, B and C). We were ultimately able to increase their stability and concentration for biophysical analyses by complex formation with viral RNA, as will be described below. Nsp1β and human PCBP2 form a complex with RNA probes that contain the slippery sequence and C-rich motif that is found within the nsp2-coding region of the PRRSV RNA genome (29Napthine S. Ling R. Finch L.K. Jones J.D. Bell S. Brierley I. Firth A.E. Protein-directed ribosomal frameshifting temporally regulates gene expression.Nat. Commun. 2017; 8 (28593994)1558210.1038/ncomms15582Crossref PubMed Scopus (53) Google Scholar). To characterize the biochemistry of this complex in greater detail, we carried out a series of EMSAs with nucleic acid probes of systematically decreasing size to identify the shortest RNA fragment to which the proteins would stably bind, with the aim of identifying a compact protein:RNA complex amenable to preparative (milligram) scale purification. It was shown previously that a 58-nt RNA probe could be used for complex formation in a native PAGE EMSA between nsp1β, PCBP2, and an extended nucleic acid probe derivative of the PRRSV (SD01-08) genome (17Napthine S. Treffers E.E. Bell S. Goodfellow I. Fang Y. Firth A.E. Snijder E.J. Brierley I. A novel role for poly(C) binding proteins in programmed ribosomal frameshifting.Nucleic Acids Res. 2016; 44 (27257056): 5491-550310.1093/nar/gkw480Crossref PubMed Scopus (29) Google Scholar). Initially, we worked with ssDNA probes (all uracil nucleotides of the frameshift site of the RNA genome changed to thymine) as DNA is more stable and cost-effective to work with. Structural studies indicate that the methyl group of thymine, which is not present on uracil, does not interact with the KH domains of PCBP2 and that it is the O2 and N3 groups of a thymine/uracil nucleoside that interact with the PCBP2 amide backbone directly (23Du Z. Lee J.K. Fenn S. Tjhen R. Stroud R.M. James T.L. X-ray crystallographic and NMR studies of protein-protein and protein-nucleic acid interactions involving the KH domains from human poly(C)-binding protein-2.RNA. 2007; 13 (17526645): 1043-105110.1261/rna.410107Crossref PubMed Scopus (39) Google Scholar, 25Fenn S. Du Z. Lee J.K. Tjhen R. Stroud R.M. James T.L. Crystal structure of the third KH domain of human poly (C) -binding protein-2 in complex with a C-rich strand of human telomeric DNA at 1. 6 Å resolution.Nucleic Acids Res. 2007; 35 (17426136): 2651-266010.1093/nar/gkm139Crossref PubMed Scopus (29) Google Scholar). Systematically, we were able to truncate the nucleic acid probe down to a minimum of 34 nt (Fig. 1B), which includes the slippery sequence, C-rich motif, and seven additional nucleotides at the 3′ end (CAGCUUU). Truncations to a size shorter than 34 nt resulted in very weak complex formation and a lack of sample monodispersity. Using the 34-nt probe, an EMSA was initially performed with WT nsp1β, PCBP2, and the ssDNA nucleic acid probe (analogous to RNA in Fig. 1B). As shown in Fig. 2B, nsp1β does not appear to interact with the nucleic acid alone even at a 20-fold molar excess in relationship to the probe, which is consistent with previous findings (17Napthine S. Treffers E.E. Bell S. Goodfellow I. Fang Y. Firth A.E. Snijder E.J. Brierley I. A novel role for poly(C) binding proteins in programmed ribosomal frameshifting.Nucleic Acids Res. 2016; 44 (27257056): 5491-550310.1093/nar/gkw480Crossref PubMed Scopus (29) Google Scholar). Interestingly, PCBP2 does interact with the ssDNA probe on its own (Fig. 2B), but not with ssRNA (Fig. 2C). This can be seen as low as an 8-fold molar excess but is highly amplified when the amount of PCBP2 is increased, as seen when a 20-fold molar excess is added in relation to nucleic acid. When both nsp1β and PCBP2 are present with the probe, a shift can be seen compared with PCBP2 bound to DNA alone, indicating the formation of a trimeric complex. Last, we wanted to confirm the importance of the cytosine-rich motif as it pertains to complex formation. The CCCATCTCC stretch of the ssDNA probe was mutated to GAAATATGG, which is termed the 34-nt CC2 DNA (14Fang Y. Treffers E.E. Li Y. Tas A. Sun Z. van der Meer Y. de Ru A.H. van Veelen P.A. Atkins J.F. Snijder E.J. Firth A.E. Efficient -2 frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein.Proc. Natl. Acad. Sci. U. S. A. 2012; 109 (23043113
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